Silk Biomaterials

2.212.

Silk Biomaterials

X Hu and D L Kaplan, Tufts University, Medford, MA, USA ã 2011 Elsevier Ltd. All rights reserved.

2.212.1. 2.212.2. 2.212.2.1. 2.212.2.2. 2.212.2.3. 2.212.3. 2.212.4. 2.212.4.1. 2.212.4.2. 2.212.4.3. 2.212.5. 2.212.5.1. 2.212.5.2. 2.212.5.3. 2.212.5.4. 2.212.6. 2.212.6.1. 2.212.6.2. 2.212.6.3. 2.212.6.4. 2.212.6.5. 2.212.6.6. 2.212.6.7. 2.212.7. 2.212.7.1. 2.212.7.2. 2.212.7.3. 2.212.7.4. 2.212.8. 2.212.8.1. 2.212.8.2. 2.212.8.3. 2.212.8.4. 2.212.9. References

Overview Silk Film Biomaterials Water Annealed and Slow Drying Silk Films Electric Field Aligned Silk Films Ultrathin Silk Films Silk Sponge Scaffold Biomaterials Silk Nanofiber Biomaterials Silk Nanofibers from Organic Solvent Silk Nanofibers from Aqueous Solvent Silk Nanofiber Composites Silk Hydrogel Biomaterials Natural Silk Hydrogel Ultrasound-Induced Silk Hydrogel Semi-interpenetrating Silk Hydrogels Injectable Silk Hydrogels Silk Microsphere and Nanoparticle Biomaterials Spray-Drying Silk Microspheres Silk Particles from Lipid–Aqueous Separation Silk Particles from Rapid Laminar Jet Silk Particles from Polymer Phase Separation Silk Particles from Organic–Aqueous Phase Separation Silk Particles from pH Variation Silk-Coated Polymer Particles Silk Optical Biomaterials Silk Nano- and Micropatterned Optical Materials Silk Optical Waveguides Color-Controllable Silk Materials Silk-Based Cornea Tissue Engineering Other Silk Materials Silk Microfluidic Devices Secondary Structure Micropatterned Silk Materials Direct-Write Silk Scaffolds Chemically Modified Silk Materials Conclusions

Glossary Biological microelectromechanical systems (BioMEMS) A special class of microelectromechanical systems (MEMS) where biological matter is manipulated to analyze and measure its activity. Electrospinning Electrospinning is a process that uses an electrical charge to draw very fine (typically on the micro or nano scale) fibers from a liquid. Layer-by-Layer (LbL) A thin film fabrication technique: the films are formed by depositing alternating layers

2.212.1.

Overview

Silks are generally defined as protein polymers that are synthesized and spun into fibers by many organisms, including

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of oppositely charged materials with wash steps in between. Spray drying A method of producing a dry powder from a liquid or slurry by rapidly drying with a hot gas. Waveguide A waveguide is a process in which a lighttransmitting material such as a glass or plastic fiber is used for transmitting information from point to point at wavelengths in the ultraviolet, visible-light or infrared portions of the spectrum. Also known as fiber waveguide/ optical-fiber cable.

silkworms, spiders, scorpions, mites, and flies.1–18 The more commonly utilized silk, such as from the silkworm, Bombyx mori, has been used in the textile industry for thousands of years due to its visual appeal and mechanical properties.9,12

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Natural silks provide structural roles in cocoon structures, web formation, traps, nest building, safety lines, and egg protection.9,12 Silks from silkworms can be obtained in significant quantities from naturally spun silk cocoons, and therefore, can be used as natural biomaterials.9–12 Silks are intriguing materials due to their combination of high toughness and strength.6,15,17 The toughness of some silks is higher than that of high-performance Kevlar fibers.15 In addition to their impressive mechanical properties, silks offer a range of useful features, including environmental stability, biocompatibility, controllable biodegradability, and morphologic flexibility.1,12,16 Thus, silks represent a unique family of structural proteins that are useful in the traditional textile industry, while also providing a wide range of properties for biomedical materials. The highly repetitive primary sequence of silk proteins leads to significant homogeneity in their secondary structures related to structural material needs, in contrast to the less ordered globular proteins that provide catalytic and molecular recognition functions.9–12 The secondary structure in silks permits tight packing of stacked beta sheets (crystalline domains) connected by hydrogen bonds between protein chains, generating hydrophobic regions with strength and resiliency.1,19–23 Therefore, silk materials have significant crystallinity and are insoluble in most solvents, including water, dilute acids, and alkali.11 The domesticated silkworm (B. mori) silk fibroin protein chain consists of two proteins: a heavy chain (
370 kDa) and a light chain (
26 kDa) which are present in a 1:1 ratio and linked by disulfide bond.5 Compared with other proteins, silks fibroins have repetitive primary structures such as (Gly-Ala-Gly-Ala-Gly-Ser)n, (Gly-Ala-Gly-Ala-Gly-Tyr)n, or AGVGYGAG motifs.5 The crystalline regions have been identified as rigid, tightly packed antiparallel beta-pleated sheets which exclude water, in part accounting for the remarkable stability of silk biomaterials.8,9 Exposure of noncrystallized silk materials to organic solvents,8,11 application of mechanical stress15,17 or thermal treatments,8,24–26 and the addition of metal ions9,10,12 among other options, all induce the irreversible crystallization of silk proteins, which has become the foundation for processing silk into biomaterials. In this chapter, we focus on recent progress in designing and producing new silk-based biomaterials, including silk film, sponges, nanofibers, hydrogels, microspheres and nanoparticles, optical biomaterials, and other systems (e.g., silk microfluidic devices, micropatterned materials, direct-write scaffolds, and chemically modified materials). The goal for this chapter is to convey the versatility and medical utility of silk-based systems to provide insight into recent silk material studies for tissue regeneration, controlled release, and biomedical optics applications and opportunities (Figure 1).

2.212.2.

Silk Film Biomaterials

Silk film formation is an efficient way to mimic the natural process of silk self-assembly and to understand the role of water during structural transitions.9,10,12,15,17 As a biomaterial, these films offer limited utility other than when used as coatings or perhaps as antiadhesion barriers. Exposure of silk films to organic acids or alcohols, applying physical stretching, or heat above the glass transition temperature induce beta-sheet

(silk II structure) crystal formation (>35%) in silk films.8,11 However, low beta-sheet content and aqueous insoluble silk films, such as alpha-helix-enriched (silk I structure) films, are difficult to produce. Therefore, many methods in the last few years have been developed to generate silk film biomaterials with low beta-sheet crystallinity for improved flexibility of films and for more rapid enzymatic degradation (Figure 2).

2.212.2.1. Water Annealed and Slow Drying Silk Films A ‘water annealing’ technique was developed to produce the low beta-sheet content silk films.27 The keys in this process are the preparation of silk films with a subsequent water-vapor annealing procedure. These films degraded more rapidly, as determined in vitro via enzymatic hydrolysis, yet supported human adult stem cell expansion in vitro in a similar or improved fashion to the high crystallinity films induced by organic solvents (e.g., methanol).27 Furthermore, slow drying at room temperature also induced the formation of insoluble silk I films.28–30 By controlling the drying rate, water-insoluble silk films can be prepared with a similar beta-sheet content as soluble silk films (
20%), versus the relatively high beta-sheet content in methanol- or water-annealed silk films (about 40 and 30%, respectively).29 The high content of silk I structure in these films resulted in more rapid degradation compared with those generated by water annealing or methanol annealing, which leads to an extended range of biomedical material applications, such as where rapid degradation in vivo is desired.29

2.212.2.2. Electric Field Aligned Silk Films Another example of new silk film biomaterials is based on the application of electric fields.31,32 By casting silk solution under an alternating electric field (AC), dramatic changes in the alignment of molecular dipoles and the formation of oriented supramolecular assemblies were achieved.32 Mechanical, thermal, and surface properties of the films were therefore changed compared to controls without this alignment process.32 Cell responses were also affected; fibroblasts cultured on these anisotropic fibroin films preferentially spread parallel to the field direction 6 h after seeding.32

2.212.2.3. Ultrathin Silk Films Compared with traditional bulk silk protein films, many advanced film techniques have been developed in recent years.33–43 One of the examples is the layer-by-layer (LbL) nanoscale thin coating technique for silk fibroin studies.33,37–43 Through an all aqueous step-wise deposition process, silk fibroin layers can be deposited on a substrate and monitored spectroscopically or with a quartz crystal microbalance.33,37–43 The silk adsorption process generated stable and reproducible layers, with control of a single layer thickness ranging from a few to tens of nanometers, determined by the concentration of silk solution, salt concentration in the dipping solution, and the method of rinsing.39–41 Compared with traditional polyelectrolyte LbL techniques, an intervening drying step is added to control the structure and stability of the self-assembled silk fibroin layers.37,42 The drying process can be used to induce beta-sheet crystal formation in the films, similar to methanol

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Figure 1 (a) Silk-based materials (B. mori silk fibroin protein); (b) secondary structure of one B. mori silk fibroin chain; (c) (Gly-Ala-Gly-Ala-Gly-Ser)n amino acid repeat units that self-assemble into antiparallel beta sheets. (b) Modified from Ha, S. W.; Gracz, H. S.; Tonelli, A. E.; Hudson, S. M. Biomacromolecules 2005, 6, 2563–2569. (c) Modified from Murphy, A. R.; John, P. S.; Kaplan, D. L. Biomaterials 2008, 29, 2829–2838.

treatment and thereby stabilizing the films to avoid dissolution in water.37,42 The assembled films were stable under physiological conditions and supported stem cell adhesion, growth, and differentiation, providing new options for engineering biomaterial coatings in medical devices as well as controlling of interfacial properties conducive to specific cellular or tissue responses.33,38,43 These LbL nanocoatings were utilized as carriers to incorporate drugs.38–41,43 Model small-molecule drugs, as well as large proteins such as azoalbumin, were incorporated into the LbL nanocoating process with ultrathin silk films. Control of

beta-sheet crystal content and the multilayer structure of the silk coatings provide control of the release properties of these incorporated compounds during in vitro studies.39–41 Higher crystallinity with thicker silk capping layers suppressed the initial burst release and prolonged the duration of release of the model drugs from the silk materials.39–41 This approach provides an important option to regulate drug release kinetics from silk by controlling its structure and morphology, which is useful in surface engineering of biomaterials and medical devices for regulating the release of drugs.39–41 The control of release kinetics of therapeutic drugs has also been validated

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Figure 2 Image of a robust, uniform, and freely suspended silk fibroin layer-by-layer (LbL) film over a 150-mm opening: (a) Interference pattern without pressure and (b) deformed silk film under a pressure of 838 Pa. Reproduced from Jiang, C. Y.; Wang, X. Y.; Gunawidjaja, R.; et al. Adv. Funct. Mater. 2007, 17, 2229–2237. (c) AFM cross-section at the film edge area to obtain thickness of a rhodamine B model drug contained LbL silk ultrathin film. Reproduced from Wang, X. Q.; Wenk, E.; Hu, X.; et al. Biomaterials 2007, 28, 4161–4169; Wang, X. Q.; Wenk, E.; Matsumoto, A.; Meinel, L.; Li, C.; Kaplan, D. L. J. Control. Release 2007, 117, 360–370; Wang, X. Y.; Hu, X.; Daley, A.; Rabotyagova, O.; Cebe, P.; Kaplan, D. L. J. Control. Release 2007, 121, 190–199.

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Figure 3 (a) Scaffolds prepared from silk fibroin in aqueous solution (8 wt%, pore size
920 mm) or hexafluoroisopropanol (HFIP) solution (8 wt%, pore size
890 mm). Scale bar ¼ 1 cm. Scanning electron microscope (SEM) images of porous scaffolds (b) prepared from silk fibroin HFIP solution (8 wt%) with NaCl particles 850–1000 mm and (c) prepared from silk fibroin aqueous solutions (8 wt%) with NaCl particles 1000–1180 mm. Reproduced from Kim, U. J.; Park, J.; Kim, H. J.; Wada, M.; Kaplan, D. L. Biomaterials 2005, 26, 2775–2785.

in vivo in epilepsy animal models with silk-based brain implants.38,43

2.212.3.

Silk Sponge Scaffold Biomaterials

Silk sponge scaffold biomaterials are frequently used systems for tissue engineering.1,12,16,44 Silk sponge scaffolds for bone and organ regeneration provide useful features such as biocompatibility, impressive mechanical properties, versatility of chemistry, aqueous processing to entrain bioactive molecules, and cell-controlled degradability.12,45 Generally, pore size and the porosity of the scaffolds are key factors to consider. Pore sizes larger than 100 mm in diameter are typically considered minimum for tissue scaffolds,45 based on cell size and migration. Porosity determines how well the scaffold pores are interconnected, which directly controls the ability

of the seeded cells to interact and signal one another.45 Depending on the applications of the scaffolds, mechanical properties as well as degradation rate are also important for supporting tissue function, integration, and growth.45 The core question during engineering silk scaffold systems is how to produce interconnected pores in a three-dimensional (3D) silk system. In addition, mechanisms to induce aqueous insolubility via beta-sheet crystallization are also required (Figure 3). At least three methods have been reported to generate porous 3D sponge matrices from silk proteins44–46: salt leaching, gas forming, and lyophilization or freeze-drying. Salt leaching utilizes porogen leaching approaches, with granular salt particles (e.g., NaCl) in silk solutions.44,45 The bulk of the silk particles are retained as solids because of saturation of the solution. Therefore, the sizes of salt particles can be used to control the pore sizes in scaffolds. Some salts (e.g., NaCl, KCl)

Silk Biomaterials

also induce beta-sheet crystals in silk protein during the process.45 After 24 h at room temperature, the insoluble silk scaffolds are formed, and the salts can then be extracted by immersion of the scaffolds in water.45 Gas forming uses bicarbonate salt (e.g., ammonium bicarbonate or sodium bicarbonate) as the porogen added to silk solution, with a porogen-to-silk weight ratio of 10:1
20:1.44 After drying and beta-sheet crystallization in alcohol, the scaffolds are immersed in 95  C water to induce gas foaming and remove/dissolve the bicarbonate particles.44 There are a variety of approaches to generate air bubbles in silk solution, after rapid crystallization by alcohol or freezing before crystallization, with the air bubbles used to generate the pores in the scaffolds.44 The freezedrying method utilizes ice particles as the source of pores.44,46 By controlling freezing rate and freezing temperature, the size of the ice particles can be controlled. Therefore, gelation and crystallizing silk solution before freeze-drying, or crystallizing silk solid scaffolds after freeze-drying, can be used to obtain insoluble silk scaffolds for tissue engineering and other biomedical applications.46 This method avoids organic solvents. Aside from the aforementioned pure silk approaches, additives such as gelatin to the silk fibroin solution can be used to control the conformation of silk with the formation of insoluble porous structures.46 The pore sizes of scaffolds were controlled by adjusting the silk fibroin concentration.46 The addition of the gelatin resulted in improved hydrophilicity and in vitro cell culture interactions compared to salt-leached silk fibroin scaffolds alone.46

2.212.4.

Silk Nanofiber Biomaterials

Nanofiber scaffolds can mimic the nanoscale properties of fibrous components in the tissue native extracellular matrix (ECM), and have been broadly explored in tissue engineering, in wound healing, and related medical applications.22,47–50

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Compared with conventional fiber-processing techniques which produce fibers with tens of microns in diameter, nanofibers are at least two orders of magnitude smaller than the conventional fibers, but with similar geometry.50 Selfassembly, phase separation, and electrospinning methods can be used to produce these types of nanofibers for different biomedical applications. Self-assembly and phase-separation techniques can generate fibers with diameters from 1 to 500 nm, but are effective with only a selected number of polymers.50 Electrospinning is a versatile technique that enables the development of nanofiber-based silk biomaterial scaffolds.50 A typical electrospinning setup usually contains three components: a high-voltage supplier, a capillary needle, and a grounded collector.50 By applying an electric potential to the droplet of protein solution suspended on the needle, repulsive forces produced by charges in the solution and the attractive forces from the collector exert tensile forces on the protein solution. A nanofiber jet can then eject from the apex of the cone and accelerates toward the grounded collector.50 Many factors can significantly affect the process of the formation of the electrospun nanofibers, including the solution conditions, such as viscosity, conductivity, concentration, surface tension, and molecular weight of the protein50; instrument impacts, such as applied electrical potential and morphology of the capillary tube50; or environmental parameters, such as temperature, humidity, and air velocity.50 If the protein solution is too dilute, the fiber may break into microsize droplets before reaching the collector, which results in the phenomenon of ‘electrospray.’ If the protein solution is too concentrated, electrospinning solution may not produce fiber due to high viscosity (Figure 4).

2.212.4.1. Silk Nanofibers from Organic Solvent Silk proteins have been widely utilized as electrospinning biomaterials. By choosing organic solvents, such as

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1 mm Figure 4 (a) Schematic of electrospinning setup; scanning electron microscope (SEM) images of crystallized and polyethylene-oxide (PEO)-extracted electrospun silk nanofibers, (b) unmodified, and (c) after adsorption of multi-wall carbon nanotubes (MWCNTs). (a) Reproduced from Zhang, X. H.; Reagan, M. R.; Kaplan, D. L. Adv. Drug Deliv. Rev. 2009, 61, 988–1006. (b and c) Reproduced from Kang, M.; Jin, H. J. Colloid Polym. Sci. 2007, 285, 1163–1167; Kang, M.; Jung, S.; Kim, H. S.; Youk, J. H.; Jin, H. J. J. Nanosci. Nanotechnol. 2007, 7, 3888–3891.

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hexafluoroacetone (HFA), hexafluoroisopropanol (HFIP), or formic acid, continuous fibers were obtained with variable mean diameters and distributions.50–52 It is believed that a faster evaporation rate of the solvent leads to the formation of thicker fibers with less elongation and lower betasheet crystal content. For example, the mean diameter of electrospun silk nanofibers dissolved in formic acid was smaller (80 nm) than those from rapidly evaporated HFIP solution (380 nm).51

2.212.4.2. Silk Nanofibers from Aqueous Solvent Wang et al.37,42,53,54 first electrospun B. mori silk fibroin using an aqueous solution, solving concerns of chemical residuals from organic solvents. Fibers formed when the aqueous silk solution reached 28% (w/v), with diameters from 400 to 800 nm.37,42,53,54 However, when electrospinning 39% (w/v) silk solution, uneven and ribbon-shaped silk fibers were observed, due to the slow water evaporation from fiber surface.37,42,53,54 Silk aqueous solutions with a viscosity lower than 40 mPa did not provide sufficient molecular chain entanglements for silk electrospinning.37,42,53,54 The pH effects on electrospun fiber morphology and properties were investigated.50 With the combined reduction in pH and concentration, the morphology of the electrospun silk fibers changed from ribbon-like to a uniform cylinder, from 265 nm in 25% (w/v) silk solution at a pH of 4.8 to 850 nm in 33% (w/v) silk solution at pH 6.9.50 Fiber diameter dependence on electric potential was also observed by Meechaisue et al.55 using 40% (w/v) silk fibroin solutions. The results indicated that when the electric field was doubled, the average diameter of electrospun silk fibers will also double.55 The major conformations in electrospun silk fibroin nanofibers when spun from aqueous solution were random coil and alpha-helix (silk I), with a little beta sheet (silk II).50 However, it is challenging to obtain highly concentrated and stable silk aqueous solutions, as self-assembly leading to gelation can occur.50,56 To solve this problem, Jin et al.56 blended poly(ethylene oxide) (PEO) with a lower concentration of aqueous silk fibroin solution. From this blend, nanofibers (
750 nm) with comparable diameters to those from pure aqueous system were generated.56 The water-soluble PEO was extracted out in distilled water after post-treatments of nanofibers to lock in the beta-sheet structure.56 There are a number of treatments commonly performed to induce insolubility of electrospun fiber matrices. Kim et al.52 used 50% (v/v) aqueous methanol for 10–60 min at room temperature. Conformational transitions of the unmodified silk fibroin nanofibers to crystalline structure were completed within 10 min.51 Owing to brittle features of these methanol-treated silk fibroin matrices, alternate methods, such as water-vapor annealing, were also developed. Jeong et al.51 reported differences in electrospun silk fibroin matrices after treatment with either aqueous methanol solution (50%) or water vapor. A longer time was required for water-vapor treatment at low temperatures, and different mechanical properties were found compared with MeOH-treated fiber mats, including better elasticity because of the lower beta-sheet content.51 The efficiency of other solvents, such as ethanol, methanol, and propanol, temperature (25–55  C) or treatment time was studied in detail.51

2.212.4.3. Silk Nanofiber Composites In addition to pure silk nanofibers, many composite electrospun nanofibrous matrices based on silk, such as silk fibroin/ chitin57 or silk fibroin/collagen58 mixtures, have been studied, using techniques such as a single-needle or side-by-side spinning approaches.50 Wang et al. successfully encapsulated a silk fibroin core fiber within a PEO shell fiber.53,54 After water-vapor treatment, PEO was extracted and the crystallized silk fibers with diameters, down to 170 nm, were obtained.53,54 Functionalized silk nanofibrous matrices have also been developed for release of molecules including antibiotics, proteins, small molecules, and DNA.50,59–62 Drugs, enzymes, growth factors, various compounds, and conductive materials can be loaded via prespinning and mixing with silk solutions, and entrained in the fibers during coaxial electrospinning or postelectrospinning by covalently coating on nanofibers.50 Lee et al. immobilized alpha-chymotrypsin (CT) on silk fibroin electrospun nanofibers with amino group preactivation with glutaraldehyde.61 A significant increase in enzyme loading was observed and the activity of the immobilized CT was almost eight times greater than that on silk microfibers.61 Li et al. incorporated bone morphogenetic protein-2 (BMP-2) into silk fibroin nanofibers in the spinning solution.62 BMP-2 encapsulated silk fibroin matrices increased calcium deposition from human bone marrow-derived mesenchymal stem cells (hMSCs) when grown on the matrices, with enhanced transcript levels of bone-specific markers.62 These BMP-2loaded electrospun silk nanofibrous matrices were efficient delivery systems to improve bone formation.62 In addition to biological functionalization, electric property modifications have also been examined in silk electrospun matrices using multiwall carbon nanotubes (MWCNTs).60,63 A significant amount of MWCNTs were retained on the surface of the nanofibers even after sonication, and the electrical conductivity of the MWCNT-absorbed silk matrices increased significantly at room temperature, compared with pure silk nanofibers.63 Future research for electrospun silk fiber may focus on combining biological assays with silk nanofiber materials to assess cellular responses.

2.212.5.

Silk Hydrogel Biomaterials

Hydrogels are insoluble 3D polymer chain networks that swell in aqueous solution and hold or entrap liquid components such as cells and drugs.21,64–67 Hydrogels exhibit solid-like mechanical behavior, with high compliance and elastic strain, while consisting mostly of liquid.21,65,66 The formation of hydrogels from solution is due to the connectivity of the protein chains as a result of ‘cross-linking’.64 Generally, there are two main types of cross-links in hydrogel systems: chemical and physical.21,66 In chemically cross-linked hydrogels, networks are formed by chemical reactions or polymerization to stitch together the starting materials (such as monomers) via cross-linkers.21,66 Physically cross-linked hydrogels can be obtained by crystallization, liquid–liquid phase separation, ionic interactions, or hydrogen bonding.21,66 Silk fibroin aqueous solutions form hydrogels by physical cross-linking, with the rate of this sol–gel transition dependent on silk

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Figure 5 (a) Typical appearance of different silk/polyacrylamide semi-interpenetrating (semi-IPNs) hydrogels, (b) scanning electron microscope (SEM) image of silk/polyacrylamide semi-IPNs with composition ratios of 70/30, and (c) Schematic illustration of mechanism of silk gelation during ultrasonication. The gelation process contains two kinetic steps: (a) structural change from random coil to beta sheet with some interchain physical cross-links occurring in a short timeframe, and (b) beta-sheet structure-extended, large quantity of interchain beta-sheet cross-links, and molecules organized to gel network over a relatively long time frame. (a–b) Reproduced from Mandal, B. B.; Kapoor, S.; Kundu, S. C. Biomaterials 2009, 30, 2826–2836. (c) Reproduced from Wang, X. Q.; Kluge, J. A.; Leisk, G. G.; Kaplan, D. L. Biomaterials 2008, 29, 1054–1064; Wang, X. Y.; Zhang, X.; Castellot, J.; Herman, I.; Iafrati, M.; Kaplan, D. L. Biomaterials 2008, 29, 894–903.

concentration, temperature, metal ions, and pH.38,43,68–70 The mechanism of gelation is self-assembly of the beta-sheet crystals.69,70 Silk hydrogels have been used for biomedical applications because of their biocompatibility and adjustable mechanical properties (Figure 5).

2.212.5.1. Natural Silk Hydrogel Early studies on the formation of silk hydrogels focused on the natural gelation process from silk fibroin aqueous solutions.69,70 The gelation time of silk hydrogels decreased with increase in protein concentration and temperature, decrease in pH, and addition of PEO or Ca2þ.69,70 However, other ions, such as Kþ, did not have a significant impact on silk gelation time.69 Freeze-dried silk hydrogel materials formed with Ca2þ exhibited larger pores than pure silk gels.69 Mechanical compressive strength and modulus of silk hydrogels can be controlled by increasing the protein concentration and gelation temperature.69 A conformational transition from random coil to beta-sheet structure promoted the formation, insolubility, and stability of silk hydrogels.68–70 The impressive mechanical properties, biocompatibility, and biodegradability of silk hydrogels prompted additional studies to improve the gelation for different biomedical applications.

2.212.5.2. Ultrasound-Induced Silk Hydrogel A novel method to accelerate the process and control silk fibroin gelation was reported through ultrasonication, as an energy input to the solution of silk to drive assembly and gel formation.38,43 Power output and the time of sonication, along with silk fibroin concentration control gelation of silk from minutes to hours, allowing the addition of cells postsonication but prior to final gel formation.38,43 hMSCs were successfully incorporated into these silk fibroin hydrogels after sonication and proliferated in the 4% silk hydrogels over 21 days.38,43

2.212.5.3. Semi-interpenetrating Silk Hydrogels Other silk-based hydrogel studies focused on producing stimuli-responsive or semi-interpenetrating (semi-IPNs) hydrogel networks by blending silk with other polymers such as gelatin71 and polyacrylamide.72 For example, silk/gelatin hydrogel systems were formed with beta sheets formed via subsequent exposure to methanol, which entangled the gelatin molecular chains and stabilized the thermally responsive hydrogel network during the structural transition of gelatin.71 Swelling and protein release kinetics of silk/gelatin hydrogels were controlled by varying composition. Through chemical cross-linking, silk/polyacrylamide hydrogels were synthesized using different ratios of silk fibroin/acrylamide mixtures.72 The properties of these semi-IPNs depended on the ratio of two components.72

2.212.5.4. Injectable Silk Hydrogels Injectable silk hydrogel systems have also been developed. By vortexing aqueous solutions of silk, a sol–gel transition was observed, with transition from random coil to beta-sheet structures, and orders of magnitude increase in shear modulus.73 These vortex-induced silk hydrogels had permanent, physical, intermolecular cross-links, and the hydrogelation kinetics could be controlled by changing vortex time, assembly temperature, and/or protein concentration.73 Such silk gel systems provide a useful time frame for cell encapsulation. The stiffness of preformed hydrogels recovered quickly, immediately after injection through a needle, enabling the use of these systems for injectable cell delivery systems.73 A gel-spinning method for silk tubes formation was developed for device formation.74 By spinning an aqueous silk solution on a tube around a reciprocating rotating mandrel, the formed silk tube biomaterial exhibited specific winding patterns, porosity, and composite features.74 Silk tube properties were further controlled via different postspinning

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processing mechanisms, such as methanol treatment, airdrying, and lyophilization, which offered numerous tissue engineering applications such as biomaterial matrices useful for blood vessel grafts and nerve guides.74 Electrogelation, the application of a low-voltage electric field to an aqueous solution of silk fibroin, generated injectable silk hydrogels with adhesive properties.75,76 This system demonstrated reversible adhesive properties and functioned on both hydrated and dry surfaces.75 The structural transition of this silk gel was found to be reversible with random coil to alpha-helix transitions present.75,76 This system utilizes all biocompatible components and functions in an all-aqueous process at ambient conditions, which provides potential applications in environmentally compatible material and medical device systems, including tissue adhesive functions.76

2.212.6. Silk Microsphere and Nanoparticle Biomaterials Microspheres (1–1000 mm) and nanoparticles (1–1000 nm) are polymer particles used for biomedical applications such as controlled drug delivery and tissue engineering.39,77–85 These structures can be used to incorporate drugs with controlled release kinetics, while retaining sufficient in vivo stability for function, biocompatibility, and degradability and the potential to target specific organs and tissues.78,79,84 Microspheres are commonly used as depot drug carriers for longacting delivery and usually administered intramuscularly or subcutaneously.84 A depot delivery system requires particle sizes above 5 mm in order to remain at the injection site and

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slowly release drug contents.84 Nanoparticles are usually designed as short-acting delivery vehicles (as a solid powder or with a liquid carrier) and administrated through intramuscular, intravenous, subcutaneous, oral, or transdermal routes.84 With a smaller size, they can penetrate through small capillaries, across physiological barriers, and become incorporated into cells for treating various diseases such as cancers.84 Silk proteins have been used to produce micro- and nanospheres.84 Compared with synthetic polymers and other natural degradable materials, silk proteins exhibit useful mechanical properties, tunable in vivo degradation rates, biocompatibility, and all aqueous material processing.39,80,82–85 For drug delivery, small-molecule drugs or protein drugs can be incorporated with high efficiency, and drug release kinetics can be modulated through the control of crystalline beta-sheet content during processing.84,86 Typical techniques available for the preparation of drug-loaded silk microsphere or nanoparticles are based on spray drying, emulsion-solvent evaporation/extraction, solvent displacement, phase separation, self-assembly, or rapid expansion of supercritical fluid solution. Silk fibroin has been fabricated into microspheres by many of these methods (Figure 6).

2.212.6.1. Spray-Drying Silk Microspheres Silk microspheres were prepared by spray-drying an aqueous solution of silk, containing theophylline as a model drug with a small amount of ethanol.87,88 The amorphous silk microspheres were then exposed to a humid atmosphere (89% relative humidity) to induce beta-sheet crystallization.87 The mean diameter of the microspheres was around 5 mm.87

(b)

Silk I

(c)

Amide I absorbance

Silk II

pH 9 pH 8 pH 7 pH 6 pH 5 pH 4 1700

1650

2 mm

2 mm

1600

(d)

(e)

5 µm

(f)

5 µm

5 µm

Figure 6 (A) FTIR spectra of silk fibroin particles produced by salting out with potassium phosphate at different pH values; (b and c) scanning electron microscope (SEM) of silk particles produced by salting out with potassium phosphate (pH 8) from a silk fibroin solution of (b) 0.25 mg ml1 and (c) 20 mg ml1; (d–f) confocal images of loading and distribution of model drugs in silk fibroin spheres prepared from silk/PVA blend solution. Model drugs: (d) tetramethylrhodamine-conjugated bovine serum albumin (TMR-BSA, MW
66 000 Da), (e) tetramethylrhodamine-conjugated dextran (TMR-dextran, MW
10 000 Da), and (f) rhodamine B (RhB, MW
479 Da) were premixed with silk solution. (a–c) Reproduced from Lammel, A. S.; Hu, X.; Park, S. H.; Kaplan, D. L.; Scheibel, T. R. Biomaterials 2010, 31, 4583–4591. (d–f) Reproduced from Wang, X. Q.; Yucel, T.; Lu, Q.; Hu, X.; Kaplan, D. L. Biomaterials 2010, 31, 1025–1035.

Silk Biomaterials

2.212.6.2. Silk Particles from Lipid–Aqueous Separation Lipid vesicles were used to encapsulate protein drugs in silk protein to form microspheres under mild processing conditions.40 Freeze-thaw treatments were applied to generate small vesicles with homogeneous size distributions.40 After lyophilization, the lipid templates were removed by methanol or sodium chloride (NaCl), and the encapsulated silk microspheres were concurrently induced to form beta-sheet crystalline structures to promote the entrapment of the protein drugs.40 The MeOH-based microspheres had an average size of 1.7 mm, while the average size of NaCl-based microspheres decreased with time of NaCl treatment, from 2.7 mm (1 h) to 1.6 mm (15 h).40

2.212.6.3. Silk Particles from Rapid Laminar Jet Large microspheres for protein drug release were fabricated using a laminar jet with aqueous silk solution,89 using a nozzle vibrating at controlled frequency and amplitude. The silk particles produced in the process had diameters in the range of 101–440 mm,89 depending on the diameter of the nozzle and the treatment to induce water insolubility of silk fibroin.

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protein concentration and mixing rate. The resulting microspheres reached an average size less than 300 nm.91 Incorporation of active ingredients into spider silk microspheres was obtained by adding a solution with the desired molecules before microsphere formation.91 A similar method was also applied to silk fibroin to form controllably sized silk microspheres.86 The engineered spider silk protein could be also assembled at an oil– water interface to form microcapsules.92 Microcapsules with sizes between 1 and 30 mm were produced by emulsifying the spider silk aqueous solution into toluene.92

2.212.6.7. Silk-Coated Polymer Particles Other than producing pure silk microspheres and nanospheres, silk proteins were also used to coat polymer particles39,40,80 such as lipid, poly(lactic-co-glycolic acid) (PLGA), and alginate microspheres, using an LbL assembly technique.39 Silk fibroin coatings stabilized microspheres from degradation, but also significantly sustained protein drug release by providing a diffusion barrier with improved mechanical strength.39 Drug release can be retarded further by controlling coating thickness and crystalline content.39

2.212.7.

Silk Optical Biomaterials

2.212.6.4. Silk Particles from Polymer Phase Separation Recently, silk microspheres and nanoparticles were produced by phase separation.82,84 For example, polyvinyl alcohol (PVA) and silk solutions were mixed and subsequently cast into films. Varying the molecular weight of PVA and the ratio between PVA and silk changed the macro- and microphase separation.84 After film dissolution in water and removal of residual PVA by subsequent centrifugation, silk spheres were recovered with a broad size distribution ranging from 300 nm up to 20 mm, with approximately 30% beta-sheet crystallinity.84

2.212.6.5. Silk Particles from Organic–Aqueous Phase Separation Using 70% (v/v) water-miscible protonic and polar aprotonic organic solvents, silk protein can form nanospheres in a size range of 35–125 nm.85,90 Silk microspheres can also be prepared via mild self-assembly of silk fibroin molecular chains by adding ethanol and quenching below the freezing point,90 as ethanol is a poor solvent for silk fibroin, but miscible with water. When a small amount of ethanol was added into the silk aqueous solution, silk fibroin molecules first formed small beta-sheet microcrystals,90 acting as a seed for the growth of silk fibroin aggregates with continuous stirring. During the freezing procedure, the amorphous phase attached or entrapped the crystalline phase to form spheres. The particles had sizes ranging from 0.2 to 1.5 mm,90 and the distribution of these silk particles was affected by the amount of ethanol, the freezing temperature, and the concentration of silk fibroin.90

2.212.6.6. Silk Particles from pH Variation For engineered spider silk proteins, microspheres were produced by salting out a high concentration of potassium phosphate at different pH.91 Sphere size and growth were controlled by

Silk is the strongest and toughest natural material known and has excellent surface flatness and optical transparency.93 Freestanding silk films, as large as 40 cm2, with a thickness between 40 and 100 mm, have excellent transparency across the visible spectrum.93 These properties are useful to generate functionalized biophotonic components in contrast to inorganic glasses and semiconductors or synthetic organic polymers, which require either chemical postprocessing or high temperatures that negatively affect biological dopants.93 Silk-based biophotonic materials have been produced recently in a variety of studies for biomedical optics. Silk-based optical elements, which offer biodegradability and biocompatibility, have led to a new class of new devices that could be used in the human body (Figure 7).93

2.212.7.1. Silk Nano- and Micropatterned Optical Materials Methods have been developed for the construction of silk fibroin-based nano- and micropatterned optical materials.93–95 This process included methods to produce optical-grade ultrapure silk fibroin solution, the casting process for patterning silk fibroin films, and the characterization of the smallest nanopatterns in silk fibroin films realized to date.93 Diffraction gratings on silk films can be formed by replicating holographic gratings with features ranging from 600 to 3600 grooves per millimeter, resulting in optical elements such as lenses, microlens arrays, or 2D diffractive optics.94 Mechanically robust, optically transparent silk films capable of sub-40 nm transverse pattern resolution were also obtained.95 By employing this technique, high-quality optical films containing intricate 2D and 3D nano- and micropatterns were fabricated,95 useful as optical elements in a range of biomedical applications. For example, silk optical elements were used as optical transducers to monitor the spectral response of the embedded biochemical compound.93

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(a) Nanopatterned silk 600 nm

500 nm

400 nm

350 nm

Nanopatterned silk + water

(b)

2 cm (c)

Figure 7 (a) Periodic imprinted nanoholes on silk films (200 nm in diameter and 30 nm deep), illuminated with a supercontinuum light source. The lattice constants vary as 600, 500, 400, and 350 nm. In the upper panel, the holes are in the air. In the bottom panel, the medium above the holes is water. Reproduced from Amsden, J. J.; Perry, H.; Boriskina, S. V.; et al. Opt. Express 2009, 17, 21271–21279. (b) Optical images of silk waveguides guiding light from a He:Ne laser source. Reproduced from Parker, S. T.; Domachuk, P.; Amsden, J.; et al. Adv. Mater. 2009, 21, 2411–2415. (c) High-quality projected image from a 3D diffraction pattern in silk fibroin film. Reproduced from Perry, H.; Gopinath, A.; Kaplan, D. L.; Negro, L. D.; Omenetto, F. G. Adv. Mater. 2008, 20, 3070–3072.

2.212.7.2. Silk Optical Waveguides Silk protein was used to generate optical waveguides through direct ink writing of pure or doped silk fibroin solution.96 The printed silk protein waveguides retain a rod-like morphology by crystallization in a methanol-rich reservoir.96 Both straight and wavy architectures of silk fibers were produced and found to guide laser light. These printed silk waveguides have potential applications for many optical devices, including implantable medical biomaterials that would resorb over time.96

2.212.7.3. Color-Controllable Silk Materials Color-controllable nanopatterns in pure silk fibroin protein films were also achieved.20,97 Periodic 2D lattices in silk films with feature sizes of hundreds of nanometers exhibited different colors as a function of varying lattice spacing.20 Further, when varying the index of refraction contrast between the nanopatterned lattice and the surrounding environment by applying liquids on top of the lattices, colorimetric shifts are observed. This feature enabled silk materials to form a new class of ‘biologically active optics,’ which can serve as a lowindex biosensor platform for integration with microfluidics and other systems as biomaterials.20,97

2.212.7.4. Silk-Based Cornea Tissue Engineering Based on the development of ‘silk optics,’ cornea tissue engineering with silk biomaterials was also studied.98,99 Silk protein films were used to replicate corneal stromal tissue architecture. The films emulated corneal collagen lamellae dimensions and were surface-patterned to guide cell alignment.98 Micropatterns with pores in 0.5–5.0 mm diameter range were introduced in the silk films to enhance translamellar diffusion of nutrients and to promote cell–cell interactions.99 Human and rabbit corneal fibroblast proliferation, alignment, and corneal ECM expression on these films in both 2D and 3D cultures were successfully demonstrated.98,99 The mechanical properties, optical clarity, and surface pattern features of these films, combined with their ability to support corneal cell functions, suggested that silk biomaterial systems offer important potential benefits for corneal tissue regeneration.

2.212.8.

Other Silk Materials

With the studies and applications of silk-based biomaterials as outlined in the prior sections, many advanced techniques and methods have been introduced in recent years. In this section, we briefly focus on several trends in producing new silk-based biomaterials, which may help to continue to foster growth with these proteins and other polymer systems (Figure 8).

2.212.8.1. Silk Microfluidic Devices Silk can be used as a biomaterial for biomicroelectrical mechanical systems (BioMEMS), due to its biocompatibility, toughness, and slow predictable biodegradation rate. Recently, techniques and materials processing strategies utilized in the fabrication of cell-seeded silk fibroin microfluidic devices were developed.23,100 Silk-based microfluidic devices promoted adhesion and function of seeded cells, and facilitated protein or growth factor incorporation under mild conditions.23 Through soft-lithographic techniques, silk microfluidic devices were fabricated. Biocompatibility and functionality of patent devices with cells was studied by seeding and perfusion with human hepatocarcinoma cells.23

2.212.8.2. Secondary Structure Micropatterned Silk Materials A technique for patterning silk secondary structure at the microscale was recently developed utilizing capillary transfer lithography (CTL).101 A sacrificial polystyrene (PS) mask was first deposited onto a flat silk film. The masked silk film was then briefly exposed to methanol vapor, which induced a localized transition to beta-sheet crystal in the exposed regions.101 After dissolving away the PS mask, a flat silk film with silk I and silk II regions alternating at the micrometer scale can be fabricated, such as a line pattern with a spacing of 10 mm or a checkerboard pattern with a spacing of 3 mm.101 The microscale silk I and silk II regions retained their intrinsic chemical and mechanical characteristics and showed welldeveloped modulation of localized properties, which have potential for tissue engineering, such as patterned silk scaffolds

Silk Biomaterials

(a)

217

(b)

1 mm O

(d)

(c)

H2N

H2N 1

2

OH

NH2 O

O

S

H2N

H2N

4

3

O OH

O

H2N 5

1

5

200 mm Figure 8 (a) Silk fibroin-based microfluidic devices (bar: 5 mm, insert picture bar: 200 mm). Reproduced from Bettinger, C. J.; Cyr, K. M.; Matsumoto, A.; Langer, R.; Borenstein, J. T.; Kaplan, D. L. Adv. Mater. 2007, 19, 2847–2850. (b) AFM force volume topography image of silk film patterned with a 3-mm checkerboard pattern. Reproduced from Gupta, M. K.; Singamaneni, S.; McConney, M.; Drummy, L. F.; Naik, R. R.; Tsukruk, V. V. Adv. Mater. 2010, 22, 115–119. (c) 3D direct ink writing of silk fibroin in liquid reservoir with a circular web shape. Reproduced from Ghosh, S.; Parker, S. T.; Wang, X. Y.; Kaplan, D. L.; Lewis, J. A. Adv. Funct. Mater. 2008, 18, 1883–1889. (d) Aniline derivatives used to modify silk (top), and live/dead fluorescent images of cells grown on carboxylic acid azosilk-1 and heptyl azosilk-5 (bottom). Reproduced from Murphy, A. R.; John, P. S.; Kaplan, D. L. Biomaterials 2008, 29, 2829–2838.

with uniform surface chemistry and variable mechanical properties or biodegradation.101 Moreover, exposing the patterned silk I/silk II film to water resulted in selective dissolution of silk I regions, which can be extended to produce micropatterned silk materials in different geometries.101 With the help of other microprinting techniques, such as inkjet printing, dip-pen lithography, nanosphere lithography, or photolithography, this method could be expanded to other surfaces with higher resolution and over larger areas.101

2.212.8.3. Direct-Write Silk Scaffolds Direct ink-writing techniques were used to fabricate 3D, microperiodic scaffolds of regenerated silk fibroin.102 Silk fibroin solution was treated as an ‘ink’ and deposited to become LbL 3D arrays of silk fibers, such as square lattice or circular webs, with diameters of individual fibers of 5 mm.102 These scaffolds contained feature sizes that were significantly smaller than those produced by other rapid prototyping techniques.102 Direct-write scaffolds used with hMSCs demonstrated support for cell adhesion and growth as well as enhanced chondrogenic differentiation.102

2.212.8.4. Chemically Modified Silk Materials Although many attempts have been made for chemically modifying silk proteins, such as cyanuric chloride-activated coupling, enzyme-catalyzed reactions with tyrosinase, or sulfation of tyrosine residues with chlorosulfonic acid,103,104 low reaction

yield and a limited variety of functional groups for modification are challenging propositions. Recently, the chemical modification of silk chains using diazonium coupling chemistry was developed to tailor the structure and overall hydrophilicity of silk fibroin protein.103,104 This reaction allows for the incorporation of a variety of functional groups using commercially available reagents. Five types of aniline derivatives, including carboxylic acid, amine, ketone, sulfonic acid, and alkyl functional groups, were used to modify the tyrosine side chains.103 The introduction of hydrophobic functional groups promoted structural conversion of the protein from random coil to beta sheet, while the addition of hydrophilic groups inhibited this process.103 When hydrophobic and hydrophilic silk derivatives were used as cell culture scaffolds, cells such as hMSCs displayed different growth rates and morphologies.103 The cells were able to attach, proliferate-differentiate, and express osteogenic markers when subjected to osteogenic stimuli, regardless of the silk chemical modification.103 These studies suggested that versatile chemistry could be widely useful for modifying silk structure and assembly, and providing new options of silk-based biomaterials.

2.212.9.

Conclusions

Silk proteins have been exploited recently in a wide range of biomaterials. By processing into a diverse set of morphologies, such as films, nanofibers, microspheres, nanoparticles, hydrogels, or different micro-/nanopatterned devices, the potential

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of silk-based biomaterials has expanded for potential biomedical applications. The versatility of silk proteins in terms of processing, including aqueous solutions, the biocompatibility, controllable in vivo biodegradation rate, along with the remarkably robust mechanical properties, prompt interest in the biomaterials generated from silk proteins. Further, the myriad of processing tools to control the structural state of silk proteins provide direct control over the mechanical properties and degradation lifetime of silk-based biomaterials. While we have emphasized more routine processing approaches, such as methanol and temperature treatments to crystallize silks, many other physical methods, such as electrical/dielectric field and electromagnetic fields, can induce beta-sheet crystallization in silk proteins, leading to new processing for silk-based biomaterials. We have also focused mainly on some of the more compelling biomaterial-related studies and applications with silk proteins, necessitating the omission of many studies and many areas of potential interest in the biomaterials field. For example, most studies on silk blending with other synthetic polymers and biomacromolecules have been omitted from this chapter.105–107

Acknowledgments The authors thank the NIH P41 Tissue Engineering Resource Center, the NSF, and the AFOSR for support of this work.

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